*3.4. Expression and Subcellular Localization of RHS in T. cruzi*

The expression of RHS in *T. cruzi* and other trypanosomes was analyzed by Western blot using anti-RHS antibodies raised against a recombinant protein carrying a 292-amino acid region from the carboxy-terminal domain of RHS (TcCLB.511055.20) of CLB. This region is conserved among RHS of some *T. cruzi* strains (Dm28c, Sylvio X10/1, Y, Bug2148, Tulahuen, TCC) and *T. cruzi marinkellei*. The location of RHS (TcCLB.511055.20) in the nucleus has been experimentally demonstrated in the nuclear subproteome of clone CLB [54].

The anti-RHS polyclonal antibodies identified different protein profiles among *T. cruzi* strains and trypanosome species. They reacted strongly with two bands of 118 kDa and 112 kDa in the *T. cruzi* clone CLB and G strain, and weakly with two additional bands of 65 kDa and 29 kDa in CLB. A single band of 65 kDa was detected in *T. cruzi marinkellei* and *T. rangeli*, and a band of 82 kDa in *T. brucei* (Figure 5A). The sizes of RHS proteins identified by Western blot are consistent with those predicted RHS ORFs in the *T. cruzi* strains and *T. cruzi marinkellei*. These results suggest that the RHS genes encoding the 118 kDa and 112 kDa proteins are expressed in the CLB and G strain, whereas the lower molecular weight (65 kDa and 29 kDa) RHS proteins are expressed only in lower amounts in CLB. *T. cruzi marinkellei* and *T. rangeli* showed a similar expression profile consisting of a single 65 kDa band. The presence of an 82 kDa RHS in *T. brucei* is in agreement with the RHS protein profile (85 to 110 kDa) described in this trypanosome [6].

**Figure 4.** Detection of potential recombination events in *T. cruzi* RHS sequences. Recombination analysis was performed using the RDP4 program composed of non-parametric recombination detection methods by the algorithms: RDP, GENECONV, MaxChi, Chimera, Bootscan, SiSscan, and 3Seq. RHS sequences of groups 1–10 (parental sequences) are highlighted in different colors and unclassified groups (recombinant sequences) are presented in black. All RHS sequences are also indicated by their access number in the TriTrypDB [41].

Permeabilized parasites were analyzed by indirect immunofluorescence, using anti-RHS antibodies (Figure 5B). Nuclear and kinetoplast DNA was labeled with DAPI, and the RHS proteins were detected with fluorescent anti-RHS antibodies (shown in blue and green, respectively). The fluorescence distribution in the permeabilized parasites is concentrated at the nuclear region, confirmed by its colocalization with DAPI (Figure 5B merge). RHS distribution was concentrated in spots within the nucleus. Anti-RHS also reacted within the nucleus of intracellular amastigote (Figure 6), but no reaction was found in trypomastigotes. Taken together, these results suggest that RHS proteins of clone CLB have a predominantly nuclear location.

μ

μ **Figure 6.** Cellular localization of RHS in the amastigote of T. cruzi. Confocal microscopy images from indirect immunofluorescence reaction with anti-RHS antibodies (diluted 1:1000) in permeabilized epimastigotes and amastigotes of clone CLB. The labeling of the nucleus and kinetoplast DNA (DAPI) and RHS proteins is shown in blue and green, respectively. Scale bar 3 µm. N, nucleus; K, kinetoplast.

#### **4. Discussion**

## *4.1. Genomic Organization and Generation of Genetic Variability in the RHS Multigene Family in T. cruzi*

′ RHS is a genus-specific multigene family identified in the genome of all trypanosomes sequenced so far. RHS genes have a retrotransposon insertion site in their 5′ coding region, which is predicted to disrupt more than 50% of the members of this family. Therefore, our phylogenetic analysis was restricted to transcribed RHS sequences with an uninterrupted ORF encoding the RHS domain. RHS proteins of clone CLB were categorized into 10 groups with significant bootstrap (Figure 2), suggesting that each RHS subfamily is a monophyletic group, as previously reported in *T. brucei* [6]. Regarding the unclassified RHS sequences, they were separated from the rest of the groups, suggesting some structural differentiation among these sequences, and they evolved together with other RHS groups. Our search showed that *T. cruzi* RHS paralogous genes shared 75–100% identity at the amino acid level, whereas they shared 30–47% identity with orthologous genes from other trypanosome species, such as *T. rangeli*, *T. grayi*, *T. evansi*, *T. vivax*, *T. brucei*, *T. theileri* and *T. conorhini*. From these results, we may infer that RHS genes evolved from a common ancestor and started diverging by speciation.

Once we defined the RHS sequence groups of *T. cruzi* CLB, the next question was whether recombination events occurred among the members of the various RHS groups including the unclassified ones. The comparison of transcribed RHS sequences showed the occurrence of one to three recombinational events resulting in a mosaic structure, which contains up to three fragments derived from different RHSs. The RHS sequences of unclassified groups comprised ~47% of total transcribed RHS, being involved in ~60% of the recombinational events in which they were used as a template to generate new RHS sequences. Our results suggest that the RHS family has been subjected to rapid gene turnover, resulting in different paralogous groups that are conserved for functional reasons. We believe that the unclassified RHSs may act as sequence reservoirs that can recombine with functional paralogs to generate diversity, and at the same time preserve intact copies in the RHS gene family. The lack of ancestral sequences could be explained by a continuous process of gene turnover mediated by gene conversion (allelic or ectopic) and unequal crossing-over.

The complexity of the RHS family may also be related to the large number of pseudogenes that comprise more than 50% of the family [2,6,7,42]. In *T. cruzi* and *T. brucei*, the repertoire of pseudogenes is of great importance in the generation of variants of multigenic families involved in parasitic virulence [6,55–59]. Taken together, these results suggest that trypanosomes developed alternative mechanisms for achieving genetic diversity in the multigene families, one of which uses incomplete genes (pseudogenes) in the generation of functional genes, while others promote recombination between functional genes. These mechanisms acting together may lead to the generation of multiple RHS sequences, resulting in the diversity within this family but preserving intact RHS copies in the genome.

Sequence diversity in the RHS multigene family of *T. cruzi* may be generated by unequal crossing-over (sister chromatid exchange and interhomolog crossover), segmental gene conversion, and interlocus nonallelic gene conversion. Tandem duplication generated by unequal crossing-over over between non-sister homologous chromatids (interhomolog crossover) may occur with the loss of tandem allelic counterparts in one of the haplotypes, leading to a condition called hemizygosity. Out of 139 transcribed RHS genes of CLB, 58 genes (~42%) have only one allele with no counterpart in the other haplotype (S or P), resulting in a hemizygous condition. We identified 22 RHS hemizygotes mapped in the subtelomere, which is a polymorphic region that is susceptible to homologous and ectopic recombination [5,45,46,49,51]. Callejas et al., 2006 [60] identified a large hemizygous subtelomere region in the chromosome I of *T. brucei*. This region accounted for three-quarters of the length of chromosome I and resulted in the amplification and divergence of gene families such as VSG (Variant Surface Glycoprotein) [60].

There is some evidence in the genome of *T. cruzi* that segmental gene conversion is involved in the generation of sequence diversity for multigene families organized in tandem array repeats [61–64]. In addition to segmental genetic conversion, we also found evidence of interlocus nonallelic gene conversion (IGC) among gene duplicates between loci. Gene conversion has been proposed as an active force in the evolution of trypanosomes [65]. Araujo et al., 2020 [66] showed that DNA replication origins in *T. cruzi* are preferentially located at the subtelomeric region, which is a site of conflict between transcription and replication that may lead to DNA double-strand breaks and generation of diversity. Wier et al., 2016 [67] suggested that gene conversion is the mechanism used by *T. brucei gambiensis* to avoid the Meselson effect of accumulation of mutations on the chromosomes for lack of sexual recombination in this species. The proposed mechanism is based on the repair of a defective gene copy on a chromosome by copying and pasting the functional gene from the homologous chromosome.

## *4.2. The Role of RHS Proteins in T. cruzi*

We found that RHS proteins are located in the nucleus of epimastigotes and amastigotes of *T. cruzi*. This is in agreement with previous work [54] that identified the presence of 74 RHS proteins with apparent molecular masses of 12 to 111 kDa in the nuclear proteome of *T. cruzi* epimastigotes [54]. These data were corroborated by Western blot analysis, in which we identified RHS proteins from 29 to 118 kDa in CLB. Despite the large number of RHSs expressed in *T. cruzi*, the profile of proteins recognized by anti-RHS antibodies is relatively simple, composed of 2–3 strongly reactive proteins. A similar profile was described in *T. brucei*, and it may be due to the absence of cross-reactivity between RHSs of different families [6].

Proteomic studies revealed that RHS proteins are expressed in epimastigotes of *T. cruzi* [68,69]. More recently, approximately 39 RHS isoforms expressed in *T. cruzi* trypomastigotes have been identified [70]. However, the diversity of RHS proteins detected by immunoblotting was more restricted, since only eight RHS isoforms were observed in this study [71]. The absence of reactivity of anti-RHS antibodies generated against the carboxy-terminal domain of RHS (TcCLB.511055.20) of CLB with *T. cruzi* trypomastigotes suggests that RHS proteins carrying the epitopes used in the mice immunization were not expressed in this developmental form. RHS proteins seem to be constitutively expressed in *T. brucei*, but they are more abundant in the procyclic forms of this parasite [6]. More recently, it has been reported that several RHSs are stage-specific regulated [10].

Since RHS is a target for the insertion of retrotransposons, the participation of RHS in controlling the expansion of these mobile elements has been proposed. Other functions for RHS have been related to *T. brucei*. TbRRM, a modulator of the chromatin structure in *T. brucei*, interacts with RHS transcripts, proteins and histones, suggesting that the RHS family could be involved in chromatin modeling [10]. Recently, it has been reported that several RHS proteins (RHS2, RHS4, and RHS6) may act as factors involved in the transcription elongation and mRNA export in *T. brucei* [11].

Little is known about the role of RHS in the *T. cruzi* life cycle. *T. cruzi* RHS proteins have been identified in the secretome of epimastigotes, trypomastigotes, and amastigotes, indicating that they are exported to the extracellular medium [71–74]. Bautista-Lopez et al., 2017 [71] showed that RHS proteins were present in the extracellular vesicles (EVs) released by *T. cruzi* trypomastigotes and amastigotes in infected Vero cells. The secreted RHS proteins reacted with sera from chronic chagasic patients ranging from asymptomatic to advanced cardiomyopathy. EVs are important modulators of the mammalian host—*T. cruzi* relationships, such as heart parasitism, susceptibility to infection of mammalian cells, and inflammatory response [72,75]. The immunoreactivity of RHSs from EVs suggests that they could participate, possibly as adjuvants, in the interaction of *T. cruzi* with the mammalian host. In this context, it is noteworthy that RHS is more abundant in the *T. cruzi* strains infective for humans (Bug2148, Y, and Sylvio X10) than in B7, which is not infective in humans [44].

In conclusion, our data suggest that unequal mitotic crossing-over and gene conversion play a significant role in shaping the patterns of homology between the RHS paralogous repeats that accelerate the generation of diversity within this multigene family. Recombination among transcribed RHS genes leads to the generation of multiple chimeric functional RHS genes. Finally, we showed the nuclear location of RHS in the replicative forms of *T. cruzi*. Although evidence for the functions of RHS in *T. cruzi* has been elusive, we suggest that these proteins could play a role in modulating the chromatin structure at the transcriptional and posttranscriptional levels, as has been suggested in *T. brucei* [10,11].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/9/1085/s1, Figure S1: Flowchart of RHS sequences' identification, and quality validation; Figure S2: Integrity and purity of RHS recombinant protein; Figure S3: Distribution of RHS sequences across the chromosomes of clone CLB of *T. cruzi*; Figure S4: Proportion of total RHS length in each chromosome of clone CLB; Figure S5: Phylogeny and classification of transcribed RHS sequences of clone CLB; Table S1. Mapping of RHS sequences on the chromosomes of clone CLB of *Trypanosoma cruzi*.

**Author Contributions:** J.F.D.S., R.T.S., A.G.C.-M., and M.M.G.T. conceived the study; W.P.B., R.T.S., E.R.F., and A.G.C.-M. designed and performed the experiments; W.P.B., R.T.S., E.R.F., A.G.C.-M., J.L.R., and J.F.D.S. contributed to the analysis and interpretation of the data; J.F.D.S. and J.L.R. wrote and edited the manuscript, M.M.G.T. and R.A.M. revised it critically; R.A.M. and J.F.D.S. were responsible for the project management and funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors wish to thank the following funding sources: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brasil, post-doctoral fellowship to E.R.F. (2016/16918-5), research thematic grant to R.A.M. and J.F.D.S. (2016/15000-4); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, master fellowship to W.P.B. (134397/2015-0), Young Talent Researcher fellowship/CNPq to R.T.S. (314048/20 13-8) and P.Q. fellowship to J.F.D.S. (306591/2015-4), R.A.M. (302068/2016-3); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, post-doctoral fellowship to A.G.C.-M. from CAPES (PROTAX). J.L.R. was supported by IDEA Plan Operativo Anual # 0012, Venezuela.

**Acknowledgments:** We thank Fernanda Sycko Uliana Marchiano e Rafaela Andrade do Carmo for assistance in the genomic analysis and with the figures.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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*Article*
